1. Technical Field
This invention relates to a protocol for the rapid determination of protein structure. In particular, the invention provides a method for rapidly obtaining protein or peptide structural information using only about 20-25% of the data set normally required in prior methods with a high degree of accuracy. The method allows the process to be automated to achieve results with a savings of time and labor.
2. Description of the Background Art
The determination of protein secondary, tertiary and quaternary structure is important in analyzing structural and functional relationships between all types of ligands and their receptors, enzymes and their substrates, or of any protein. For example, protein structure determination of a particular receptor generally can assist in rational drug design efforts to discover or synthesize more potent ligands at that receptor, or to design ligands with different activity profiles. Thus, existing pharmaceutical agents may be improved or changed to alter activity using detailed protein structural information. In addition, new chemical agents useful for treating disease can be developed using detailed structural information about receptors or other proteins in solution, receptors bound with ligand, or both.
X-ray crystallography is widely used to obtain detailed structural information about proteins and can provide the complete tertiary structure (global fold) of the backbone of a protein. This method, however, has several disadvantages. For example, only proteins which can be crystalized may be studied using X-ray crystallography. Some proteins are very difficult or impossible to crystalize. Moreover, crystalization can be very time consuming and expensive. Another major disadvantage of this method is that the structural information obtained is pertinent to the crystalline structure of the protein rather than the structure of the protein in solution. The bond angles present in a crystal structure may not be the same as those of the protein when it is in its active conformation and therefore may not provide information relevant to the biological or physiological system of interest.
Protein structure determination by high resolution multinuclear NMR also has become well known. In principle, this method gives all the information needed to determine the structure of a protein. Practically, however, the method is extremely time-consuming. In addition, in the past it has been very difficult to obtain accurate information about the structure of large proteins, for example 30-40 kilodaltons and especially 50 kilodaltons or larger using this method.
Traditional methods for the determination of protein structure using NMR utilized distance data derived from NOE spectra. Very recently, residual dipolar couplings have become established as additional conformational restraints in the determination of the solution structures of proteins via high resolution multinuclear NMR. Tolman et al., Proc. Natl. Acad. Sci. USA 92:9270-9283, 1995; Tjandra et al., J. Am. Chem. Soc. 118:6264-6272, 1996; Tjandra and Bax, Science 278:1111-1114, 1997; Bax and Tjandra, J. Biomol. NMR 10:289-292. The introduction of a number of lyotropic dilute liquid-crystalline solutions and other methods for weak macromolecular alignment has enabled straightforward measurement of these couplings for a variety of macromolecules. See Bax and Tjandra, J. Biomol. NMR 10:289-292, 1997; Losonczi et al., J. Biomol. NMR 12:447-451, 1998; Prosser et al., J. Am. Chem. Soc. 120:11010-11011, 1998; Clore et al., J. Am. Chem. Soc. 120:10571-10572, 1998; Hansen et al., Nature Str. Biol. 5:1065-1074, 1998; Kiddle and Homans, FEBS Lett. 436:128-130, 1998; Wang et al., J. Biomol. NMR 12:443-446, 1998; Ottinger and Bax, J. Biomol. NMR 13:187-191, 1999; Fleming et al., J. Am. Chem. Soc. 122:5224-5225, 2000; Ruckert and Otting, J. Am. Chem. Soc. 122:7793-7797, 2000.
Recently, interest has developed in the rapid determination of protein structural information based on residual dipolar couplings. Mueller et al. have developed a methodology for orienting peptide planes using dipolar couplings which determined the global fold of maltose binding protein in complex with β-cyclodextrin. This gave rise to pairwise RMSD (root mean square deviation) values between N- and C-terminal domains of the NMR structure and the corresponding regions in the X-ray structure of 2.8 Å and 3.1 Å, respectively. Mueller et al., J. Mol. Biol. 300:197-212, 2000; Mueller et al., J. Biomol. NMR 18:183-188, 2000. Lower values indicate less variation in the calculations and a more accurate structure. Generally, any value greater than 3 Å is considered quite inaccurate. Therefore, improvements in the variation would be greatly desired.
Fowler et al. (J. Mol. Biol. 304:447-460, 2000) have utilized Ni—HiN, HiN—Hαi, HiN—HNαi±1, HiN—HNi+1 resisual dipolar couplings together with a small number of backbone-sidechain NOEs to determine the backbone fold of acyl carrier protein to an RMSD between backbone atoms of about 3 Å. Hus et al. (J. Mol. Biol. 298:927-936, 2000) have utilized long-range order restraints available from paramagnetic systems in combination with residual dipolar couplings to define the fold of cytochrome C′ in the complete absence of NOE restraints. Very recently, this same group has determined the global fold of ubiquitin to 1.0 Å backbone RMSD (residues 1-71) with respect to the solution structure determined by conventional methods, using restraints derived solely from Ni+1—Hi+1N, C′i—Ni+1, C′i—Hi+1N, Ciα—C′i, Cα—Hα and Cα—Cβ residual dipolar couplings in two independent tensor frames. Hus et al., J. Am. Chem. Soc. 123:1541-1542, 2001.
These methods of protein fold determination, however effective, have the major drawback of being difficult and time-consuming. Furthermore, the complexity of the calculations needed and the large number of data points makes determination of the global fold of large proteins difficult to obtain. This is due largely to the use of universally isotopically enriched material which yield split signals in the NMR spectrum, each of which need to be assigned before a structure determination can be commenced. Splitting of signals results in both more and weaker signals. This phenomenon causes overlap of signals and a far inferior signal-to-noise ratio, both of which make the assignment process more difficult and rule out automation of the process. Methods currently available therefore can provide accurate structures or provide some structural data relatively quickly and easily, but no methods for rapid determination of the global fold are available which can also achieve the degree of accuracy which is desired. The ability to automate the various steps in the process would be of great advantage in achieving both rapid and sufficiently accurate results, however this has not been possible using the available techniques.